In Vitro Assembly and Analysis of the Apoptosome Complex
| Autor: | Marion MacFarlane, Claudia Langlais, Kelvin Cain, Michelle A. Hughes |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
medicine.diagnostic_test
Tetrapeptide biology Cell-Free System Blotting Western General Biochemistry Genetics and Molecular Biology In vitro Cell-free system Cell Line Rats Blot Western blot Biochemistry Apoptosomes Caspases medicine biology.protein Chromatography Gel Bioassay Animals Humans Fluorometry Apoptosome Caspase |
| Zdroj: | Cold Spring Harbor protocols. 2015(12) |
| ISSN: | 1559-6095 |
| Popis: | This protocol describes an in vitro model for studying the mechanisms of caspase activation and native apoptosome complex assembly in cell-free extracts. Active caspases in dATP-activated lysates are detected by fluorimetry using a tetrapeptide substrate (DEVD) tagged with a fluorophore (AFC), which, when released, produces a real-time readout for caspase-3 and -7 (DEVDase) activity. Gel filtration is used to isolate the apoptosome complex from the activated lysates, and assembly of Apaf-1 and caspase-9 from their monomeric forms into the multiprotein apoptosome can be confirmed via western blot. Apoptosome complex activity can be shown by incubation with exogenous procaspase-3 and -7 followed by fluorimetric bioassay (to confirm functionality of the processed effector caspases) and/or western blotting (for detection of cleaved caspase-3 and -7). A method for preparation of free procaspases for the bioassay is also described. |
| Databáze: | OpenAIRE |
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